Integrated nutrient recycling: Ammonia recovery from thermophilic composting of shrimp aquaculture sludge via self-heated bench-scale reactor and mango plant growth enhancement by the compost.

Due to the rapid growth of the aquaculture industry, large amounts of organic waste are released into nature and polluted the environment. Traditional organic waste treatment such as composting is a time-consuming process that retains the ammonia (NH3) in the compost, and the compost produced has little economic value as organic fertilizer. Illegal direct discharge into the environment is therefore widespread. This study investigates the recovery of NH3 through thermophilic composting of shrimp aquaculture sludge (SAS) and its application as a soil conditioner for the growth of mango plants. A maximum composting temperature of 57.10 °C was achieved through self-heating in a 200 L bench-scale reactor, resulting in NH3 recovery of 224.04 mol/ton-ds after 14 days. The addition of calcium hydroxide and increased aeration have been shown to increase NH3 volatilization. The recovered NH3 up to 3 kg-N can be used as a source of clean nitrogen for high-value microalgae cultivation, with a theoretical yield of up to 34.85 kg-algae of microalgae biomass from 1 ton-ds of SAS composting. Despite the high salinity, SAS compost improved mango plant growth and disease resistance. These results highlight the potential of SAS compost as a sustainable source of clean nitrogen for microalgae cultivation and soil conditioner, contributing to a waste-free circular economy through nutrient recycling and sustainable agriculture.

The effect of calcium hydroxide addition on enhancing ammonia recovery during thermophilic composting in a self-heated pilot-scale reactor.

A modified outdoor large-scale nutrient recycling system was developed to compost organic sludge and aimed to recover clean nitrogen for the cultivation of high-value-added microalgae. This study investigated the effect of calcium hydroxide addition on enhancing NH3 recovery in a pilot-scale reactor self-heated by metabolic heat of microorganisms during thermophilic composting of dewatered cow dung. 350 kg-ww of compost was prepared at the ratio of 5: 14: 1 (dewatered cowdung: rice husk: compost-seed) in a 4 m3 cylindrical rotary drum composting reactor for 14 days of aerated composting. High compost temperature up to 67 °C was observed from day 1 of composting, proving that thermophilic composting was achieved through the self-heating process. The temperature of compost increases as microbial activity increases and temperature decreases as organic matter decreases. The high CO2 evolution rate on day 0-2 (0.02-0.08 mol/min) indicated that microorganisms are most active in degrading organic matter. The increasing conversion of carbon demonstrated that organic carbon was degraded by microbial activity and emitted as CO2. The nitrogen mass balance revealed that adding calcium hydroxide to the compost and increasing the aeration rate on day 3 volatilized 9.83 % of the remaining ammonium ions in the compost, thereby improving the ammonia recovery. Moreover, Geobacillus was found to be the most dominant bacteria under elevated temperature that functions in the hydrolysis of non-dissolved nitrogen for better NH3 recovery. The presented results show that by thermophilic composting 1 ton-ds of dewatered cowdung for NH3 recovery, up to 11.54 kg-ds of microalgae can be produced.

Effect of oxygen deficiency on organic matter decomposition during the early stage of composting.

This study aimed to elucidate the recovery of organic matter decomposition after oxygen deficiency in the early stage was replaced by aerobic conditions during composting. Oxygen deficiency at the early stage was created by supplying nitrogen gas into the composting for 3 days (LN3dA) and 5 days (LN5dA). Subsequently, air was introduced until the end of composting instead of nitrogen gas. Runs LN3dA and LN5dA had lower organic matter decomposition by 10% and 19%, respectively, compared with fully aerobic composting (LA) after oxygen deficiency was changed to aerobic conditions. Compared with fully aerobic composting, composting with oxygen deficiency at the early thermophilic stage had a different bacterial community, as analyzed by high-throughput sequencing. During vigorous organic matter decomposition, Bacillus was dominant in Run LA, whereas Caldibacillus proliferated in Runs LN3dA and LN5dA. Bacillus thermoamylovorans, Bacillus arbutinivorans, and Bacillus kokeshiiformis were hypothesized to be inhibited by Caldibacillus. Moreover, dissimilarity analysis indicated that different bacterial communities remained until the end of composting, which could be a reason for the incomplete recovery of organic matter decomposition. As analyzed by the API-ZYM kit, the enzymatic activities were also different between all composting runs. One of the characterized enzymes, α-galactosidase, displayed low activity during oxygen deficiency and could not achieve high activity with sufficient oxygen until composting was completed. Overall, our study showed that oxygen deficiency at the early thermophilic stage caused incomplete recovery of organic matter decomposition.

Complexity of acclimatization substrate affects anaerobic digester microbial community response to organic load shocks.

This study elucidated the changes in the short-term response to organic load shocks of the anaerobic digestion (AD) microbiome acclimatized to a simple substrate and a complex substrate. Batch vial reactors were inoculated with AD sludge acclimatized to either a simple (starch and hipolypeptone) or a complex (dog food and starch) substrate, both with carbon-to-nitrogen ratio of 25. Organic loads in the form of an easily degradable substrate mix (starch and hipolypeptone) with concentrations varying from 0 to 5 g VS/L were applied to the reactors. Runs utilizing the inoculum acclimatized to a complex substrate sustained its methane productivity despite the high organic load shocks which the inoculum acclimatized to a simple substrate was unable to handle efficiently. The alpha-diversity of the microbiome decreased with increase in organic load for inoculum acclimatized with a simple substrate but was unaffected for the case of the inoculum acclimatized with a complex substrate. LactobacillalesandCloacimonadales were inferred to be major players in starch degradation pathways for the inoculum acclimatized using a simple substrate as predicted by the bioinformatics package PICRUSt2. However, acclimatizing using a complex substrate did not support their growth and were replaced by Coriobacteriales which provided higher flexibility in terms of the predicted regulated metabolic functions. The predicted functional regulation of Synergistales and Syntrophales increased with acclimatization using a complex substrate which also showed increase in the flexibility of the microbiome towards handling organic load shocks. Acetoclastic pathway was upregulated with increase in organic load regardless of the acclimatization substrate while the hydrogenotrophic pathway was downregulated. Overall, acclimatization using a complex substrate increased the robustness and flexibility of the microbiome towards organic load shocks.

Effects of oxygen supply rate on organic matter decomposition and microbial communities during composting in a controlled lab-scale composting system.

The study aimed to elucidate the effect of oxygen supply rate (OSR) on the composting of model organic waste independently from other factors by using a controlled laboratory-scale reactor system. Four OSRs, 96.2, 24.2, 13.7, and 3.45 mL-O2/min/kg-WS (wet solid), were tested. The delay of organic matter degradation was observed temporarily in the early stage of composting with 13.7 mL-O2/min/kg-WS and severe oxygen deficiency was observed in lower OSR, indicating that the critical OSR existed around this value. Composting with 3.45 mL-O2/min/kg-WS resulted in constantly low CO2 evolution rate and remarkably low degree of organic matter degradation. Under deficient oxygen, all enzymes measured, such as phosphatases, esterases, lipases, proteases, and sugar degrading enzymes, showed lower activities. High-throughput sequencing revealed Caldibacillus and Ureibacillus became dominant in the later stages of the oxygen deficiency composting, while Geobacillus was the most dominant microorganism throughout composting with OSR higher than 13.7 mL-O2/min/kg-WS.

Co-occurrence network analysis reveals loss of microbial interactions in anaerobic digester subjected to repeated organic load shocks.

Fluctuations in the anaerobic digestion (AD) organic loading rate (OLR) cause shocks to the AD microbiome, which lead to unstable methane productivity. Managing these fluctuations requires a larger digester, which is impractical for community-scale applications, limiting the potential of AD in advancing a circular economy. To allow operation of small-scale AD while managing OLR fluctuations, we need to tackle the issue through elucidation of the microbial community dynamics via 16S rRNA gene sequencing. This study elucidated the interrelation of the AD performance and the dynamics of the microbial interactions within its microbiome in response to repeated high OLR shocks at different frequencies. The OLR shocks were equivalent to 4 times the baseline OLR of 2 g VS/L/d. We found that less frequent organic load shocks result to deterioration of methane productivity. Co-occurrence network analysis shows that this coincides with the breakdown of the microbiome network structure. This suggests loss of microbial interactions necessary in maintaining stable AD. Identification of species influencing the network structure revealed that a species under the genus Anaerovorax has the greatest influence, while orders Spirochaetales and Synergistales represent the greatest number of the influential species. We inferred that the impact imposed by the OLR shocks shifted the microbiome activity towards biochemical pathways that are not contributing to methane production. Establishing a small-scale AD system that permits OLR fluctuations would require developing an AD microbiome resilient to infrequent organic loading shocks.

Effect of hydrothermal treatment on organic matter degradation, phytotoxicity, and microbial communities in model food waste composting.

Hydrothermal treatment (HTT) as a pretreatment method for compost raw material has multiple benefits such as enhanced solubility of organic material, improved bioaugmentation, and reduced biohazard by killing harmful microorganisms. In this study, we pretreated food waste via HTT at 180 °C for 30 min to investigate its effect on food waste composting. HTT generated 8.98 mg/g-dry solid (g-ds) of 5-hydroxymethylfurfural and 4.32 mg/g-ds furfural. These furan compounds were completely decomposed in the early stage of composting, subsequently the organic matter in the food waste started to be degraded. The HTT-pretreated experiment demonstrated less organic matter degradation during composting as well as lower compost phytotoxicity compared to the non-HTT-pretreated experiment, where the conversion of carbon was 25.2% and the germination index value was 55%. HTT probably denatured part of the organic matter and making it more difficult to decompose, thereby preventing the rapid release of high concentrations of phytotoxic compounds such as organic acids and ammonium ions during composting. High-throughput microbial community analysis revealed that only Firmicutes appeared in the HTT-pretreated experiment, however, other bacterial groups also appeared in the non-HTT-pretreated experiment. This was possibly influenced by furan compounds and the changes of easily degradable organic matter to hardly degradable. Bacillus and Lysinibacillus were dominant in both composting experiments during vigorous organic matter degradation, suggesting that these bacterial groups were the main contributors to food waste composting. This study suggests that HTT is advantageous for the pretreatment of easily degradable food waste, as compost with less phytotoxicity was produced.

Inoculation of Neurospora sp. for improving ammonia production during thermophilic composting of organic sludge.

Recent attempts have been made to develop a thermophilic composting process for organic sludge to not only produce organic fertilizers and soil conditioners, but to also utilize the generated ammonia gas to produce high value-added algae. The hydrolysis of organic nitrogen in sludge is a bottleneck in ammonia conversion, and its improvement is a major challenge. The present study aimed to elucidate the effects of inoculated Neurospora sp. on organic matter decomposition and ammonia conversion during thermophilic composting of two organic sludge types: anaerobic digestion sludge and shrimp pond sludge. A laboratory-scale sludge composting experiment was conducted with a 6-day pretreatment period at 30 °C with Neurospora sp., followed by a 10-day thermophilic composting period at 50 °C by inoculating the bacterial community. The final organic matter decomposition was significantly higher in the sludge pretreated with Neurospora sp. than in the untreated sludge. Correspondingly, the amount of non-dissolved nitrogen was also markedly reduced by pretreatment, and the ammonia conversion rate was notably improved. Five enzymes exhibiting high activity only during the pretreatment period were identified, while no or low activity was observed during the subsequent thermophilic composting period, suggesting the involvement of these enzymes in the degradation of hardly degradable fractions, such as bacterial cells. The bacterial community analysis and its function prediction suggested the contribution of Bacillaceae in the degradation of easily degradable organic matter, but the entire bacterial community was highly incapable in degrading the hardly degradable fraction. To conclude, this study is the first to demonstrate that Neurospora sp. decomposes those organic nitrogen fractions that require a long time to be decomposed by the bacterial community during thermophilic composting.

Effect of enzymatic pre-treatment on thermophilic composting of shrimp pond sludge to improve ammonia recovery.

In recent years, attempts have been made to develop a thermophilic composting process for organic sludge to produce ammonia gas for high value-added algal production. However, the hydrolysis of non-dissolved organic nitrogen in sludge is a bottleneck for ammonia conversion. The aim of this study was to identify enzymes that enhance sludge hydrolysis in a thermophilic composting system for ammonia recovery from shrimp pond sludge. This was achieved by screening useful enzymes to degrade non-dissolved nitrogen and subsequently investigating their effectiveness in lab-scale composting systems. Among the four hydrolytic enzyme classes assessed (lysozyme, protease, phospholipase, and collagenase), proteases from Streptomyces griseus were the most effective at hydrolysing non-dissolved nitrogen in the sludge. After composting sludge pre-treated with proteases, the final amount of non-dissolved nitrogen was 46.2% of the total N in the control sample and 22.3% of the total N in the protease sample, thus increasing the ammonia (gaseous and in-compost) conversion efficiency from 41.5% to 56.4% of the total N. The decrease in non-dissolved nitrogen was greater in the protease sample than in the control sample during the pre-treatment period, and no difference was observed during the subsequent composting period. These results suggest that Streptomyces proteases hydrolyse the organic nitrogen fraction, which cannot be degraded by the bacterial community in the compost. Functional potential analysis of the bacterial community using PICRUSt2 suggested that 4 (EC:3.4.21.80, EC:3.4.21.81, EC:3.4.21.82, and EC:3.4.24.77) out of 13 endopeptidase genes in S. griseus were largely absent in the compost bacterial community and that they play a key role in the hydrolysis of non-dissolved nitrogen. This is the first study to identify the enzymes that enhance the hydrolysis of shrimp pond sludge and to show that the thermophilic bacterial community involved in composting has a low ability to secrete these enzymes.

Short-term changes in the anaerobic digestion microbiome and biochemical pathways with changes in organic load.

Fluctuations in organic loading rate are frequently experienced in practical-scale anaerobic digestion systems. These impose shocks to the microbiome leading to process instability and failure. This study elucidated the short-term changes in biochemical pathways and the contributions of microbial groups involved in anaerobic digestion with varying organic load shocks. A mixture of starch and hipolypeptone corresponding to a carbon-to­nitrogen ratio of 25 was used as substrate. Batch vial reactors were run using acclimatized sludge fed with organic load varying from 0 to 5 g VS/L. Methane yield decreased with increasing organic load. The microbiome alpha diversity represented as the number of operational taxonomic units (OTUs) and the Shannon index both decreased with organic load indicating microbiome specialization. The biochemical pathways predicted using PICRUSt2 were analyzed along with the corresponding contributions of microbial groups leading to a proposed pathway of substrate utilization. Genus Trichococcus (order Lactobacillales) increased in contribution to starch degradation pathways with increase in organic load while genus Macellibacteroides (order Bacteroidales) was prominent in contribution to bacterial anaerobic digestion pathways. Strictly acetoclastic Methanosaeta increased in prominence over hydrogenotrophic Methanolinea with increase in organic load. Results from this study provide better understanding of how anaerobic digesters respond to organic load shocks.

Tolerance of Tetraselmis tetrathele to High Ammonium Nitrogen and Its Effect on Growth Rate, Carotenoid, and Fatty Acids Productivity.

Microalgae can use either ammonium or nitrate for its growth and vitality. However, at a certain level of concentration, ammonium nitrogen exhibits toxicity which consequently can inhibit microalgae productivity. Therefore, this study is aimed to investigate the tolerance of Tetraselmis tetrathele to high ammonium nitrogen concentrations and its effects on growth rate, photosynthetic efficiency (F v /F m ), pigment contents (chlorophyll a, lutein, neoxanthin, and ß-carotene), and fatty acids production. Experiments were performed at different ammonium nitrogen concentrations (0.31-0.87 gL-1) for 6 days under a light source with an intensity of 300 µmol photons m-2 s-1 and nitrate-nitrogen source as the experimental control. The findings indicated no apparent enhancement of photosynthetic efficiency (F v/F m) at high levels of ammonium nitrogen ( NH 4 + -N) for T. tetrathele within 24 h. However, after 24 h, the photosynthetic efficiency of T. tetrathele increased significantly (p < 0.05) in high concentration of NH 4 + -N. Chlorophyll a content in T. tetrathele grown in all of the different NH 4 + -N levels increased significantly compared to nitrate-nitrogen (NO3-N) treatment (p < 0.05); which supported that this microalgal could grow even in high level of NH 4 + -N concentrations. The findings also indicated that T. tetrathele is highly resistant to high ammonium nitrogen which suggests T. tetrathele to be used in the aquaculture industry for bioremediation purpose to remove ammonium nitrogen, thus reducing the production cost while improving the water quality.

Characterizing the microbial community involved in anaerobic digestion of lipid-rich wastewater to produce methane gas.

This study attempted to characterize the microbial community and its role in anaerobic digestion of lipid. Reactors were fed semi-continuously with three related substrates, oil and its degradation intermediates (glycerol and long chain fatty acids (LCFAs)), with a stepwise increase in organic loading rate for 90 days. Microbial community analysis using next-generation sequencing (NGS) with the MiSeq Illumina platform revealed that Anaerolineaceae was the most dominant group of bacteria in all experiments, whereas Clostridium, Desulfovibrio, Rikenellaceae, and Treponema were observed characteristically in glycerol degradation and Leptospirales, Synergistaceae, Thermobaculaceae and Syntrophaceae were seen with high abundance in LCFA and oil mineralization. Furthermore, it was discovered that Methanosaeta was the most dominant archaea. The role of these microorganisms in the methane production from oil was estimated by comparing the microbial groups in the fermentation using three substrates, and a hypothetical pathway of the methane production was proposed.

Changes in the microbial community during the acclimation process of anaerobic digestion for treatment of synthetic lipid-rich wastewater.

Changes in the microbial community were investigated during the acclimation process of anaerobic digestion while treating synthetic lipid-rich wastewater, which comprised of glucose, acetic acid, lactic acid, and soybean oil. The oil content in the synthetic wastewater was increased successively from 0% to 25% and finally to 50% of the total carbon content, to clarify the effect of substrate type change from easily degradable organic materials to lipid. The oil decomposition-associated methane production rate increased as the microorganisms acclimated to the oil and eventually levelled off around 0.76 L/d. Analysis of the microbial community using next generation 16S rRNA gene sequencing (NGS) revealed the characteristic changes of dominant microorganisms Synergistales, Anaerolineales, Actinomycetales, and Nitrospirales from the domain bacteria, and Methanobacteriales and Methanosarcinales from the domain archaea. The increase in the relative abundance of Synergistales was found to be highly correlated with the increased rate of methane production from oil.

Lactic acid bacteria modulate organic acid production during early stages of food waste composting.

Lactic acid bacteria are observed during early stages of almost all food waste composting. Among them, 2 types of lactic acid bacteria, Pediococcus (homofermentative lactic acid bacterium) and Weissella (heterofermentative lactic acid bacterium) have been often reported. In this study, the roles of these 2 types of lactic acid bacteria in the composting were tried to elucidate. It has been pointed out that Pediococcus accelerates the composting process by producing lactic acid which prevented acetic acid generation, thus activating indigenous composting microorganisms. On the other hand, this study elucidated that Weissella produced acetic acid of 20 mg g-1 DS, which is harmful to composting microorganisms, resulting in the inhibition of vigorous organic matter degradation. When these 2 coexist in the starting material, whether the composting succceeds or not depends on the ratio of these 2 lactic acid bacteria. If Pediococcus and Weissella ratio was higher than 101.5, acetic acid level was almost 3 times lower than that observed in the composting with their lower ratios of 1 and 10-1, probably because of the interaction of Pediococcus and Weissella resulting in the suppression of Weissella activity, and thus composting was accelerated.

Effect of Ca(OH)2 dosing on thermophilic composting of anaerobic sludge to improve the NH3 recovery.

The primary biological treatment method for organic sludge is composting and/or anaerobic digestion, but their product (compost or biogas) is of little economic benefit; therefore, an improved process to produce a high-value product is required to make sludge management more sustainable. Maximizing NH3 gas recovery during composting processes has the potential benefit of producing high-value microalgal biomass. However, the majority of produced ammonia does not evaporate as NH3 gas but retains as NH4+-N in the compost after fermentation. The present study investigates the effects of the timing of Ca(OH)2 dosing (on days 2, 5, and 9), and the Ca(OH)2 dose (1.1-2.6 mmol/batch), on lab-scale thermophilic composting of anaerobic sludge. The effects on NH3 recovery, organic matter degradability, and microbial activity are evaluated. Ca(OH)2 dosing immediately improved the emission of NH3, with yields 50-69% higher than those under control conditions. The timing of the dosing did not influence NH3 recovery or organic matter degradability. Higher Ca(OH)2 doses resulted in higher NH3 recovery, while microbial activity was temporarily and marginally inhibited. The pH of the compost reached 10-11.5 but quickly dropped to 8-8.5 within a day, probably because of neutralization of Ca(OH)2 by the emitted CO2 and release of NH3, which maintained the microbial activity. The present study indicated that Ca(OH)2 dosing would be useful to apply during thermophilic composting for NH3 recovery to cultivate high-value microalgal biomass, which enables this process to obtain a more economic benefit.

Enhanced fermentative hydrogen production from industrial wastewater using mixed culture bacteria incorporated with iron, nickel, and zinc-based nanoparticles.

The present study assessed the efficiency of utilizing mixed culture bacteria (MCB) incorporated with individual nanoparticles (NPs), i.e., hematite (α-Fe2O3), nickel oxide (NiO), and zinc oxide (ZnO), dual NPs (α-Fe2O3 + NiO, α-Fe2O3 + ZnO, and NiO + ZnO), and multi-NPs (α-Fe2O3 + NiO + ZnO) for hydrogen production (HP) from industrial wastewater containing mono-ethylene glycol (MEG). When MCB was individually supplemented with α-Fe2O3 (200 mg/L), NiO (20 mg/L), and ZnO NPs (10 mg/L), HP improved significantly by 41, 30, and 29%, respectively. Further, key enzymes associated with MEG metabolism, such as alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and hydrogenase (hyd), were rapidly and substantially enhanced in the medium. NiO and ZnO NPs notably promoted ADH and ALDH activities, respectively, while α-Fe2O3 exhibited superior impact on hyd activity. Maximum hydrogen production rate was concomitant with higher acetic acid production and lower residual acetaldehyde and ethanol. HP using MCB supplemented with individual NiO (20 mg/L) and ZnO NPs (10 mg/L) further improved by 8.0%-14% when dual and multi-NPs were used; the highest HP was recorded when multi-NPs were used. In addition, NPs incorporation resulted in substantial increase in the relative abundance of Clostridiales (belonging to family Clostridiaceae; > 83%). Overall, this study provides significant insights into the impact of NPs on hydrogen production from MEG-contaminated wastewater.

Effect of temperature on thermophilic composting of aquaculture sludge: NH3 recovery, nitrogen mass balance, and microbial community dynamics.

Development of thermophilic composting for maximizing NH3 gas recovery would enable the production of a nitrogen source which is free from pathogen/heavy metal, for the cultivation of high-value microalgae. The present study examined the effect of NH3 recovery, nitrogen mass balance, and microbial community dynamics on thermophilic composting of shrimp aquaculture sludge. The emission of NH3 gas at 60 and 70 °C was 14.7% and 15.6%, respectively, which was higher than that at 50 °C (9.0%). The nitrogen mass balance analysis revealed that higher temperatures enhanced the solubilization of non-dissolved nitrogen and liberation of NH3 gas from the produced NH4+-N. High-throughput microbial community analysis revealed the shift of the dominant bacterial group from Bacillus to Geobacillus with the rise of composting temperature. In conclusion, thermophilic composting of shrimp aquaculture sludge at 60-70 °C was the most favorable condition for enhancing NH3 gas recovery.

Application of Bacillus sp. TAT105 to reduce ammonia emissions during pilot-scale composting of swine manure.

Thermophilic ammonium-tolerant bacterium Bacillus sp. TAT105 grows and reduces ammonia (NH3) emissions by assimilating ammonium nitrogen during composting of swine feces. To evaluate the efficacy of a biological additive containing TAT105 at reducing NH3 emissions, composting tests of swine manure on a pilot scale (1.8 m3) were conducted. In the TAT105-added treatment, NH3 emissions and nitrogen loss were lower than those in the control treatment without TAT105. No significant difference was detected in losses in the weight and volatile solids between the treatments. Concentration of thermophilic ammonium-tolerant bacteria in the compost increased in both treatments at the initial stage of composting. In the TAT105-added treatment, bacterial concentration reached ~109 colony-forming units per gram of dry matter, several-fold higher than that in the control and stayed at the same level until the end. These results suggest that TAT105 grows during composting and reduces NH3 emissions in TAT105-added treatment.

Temperature control strategy to enhance the activity of yeast inoculated into compost raw material for accelerated composting.

The effects of inoculating the mesophilic yeast Pichia kudriavzevii RB1, which is able to degrade organic acids, on organic matter degradation in composting were elucidated. When model food waste with high carbohydrate content (C/N=22.3) was used, fluctuation in the inoculated yeast cell density was observed, as well as fluctuation in the composting temperature until day 5 when the temperature rose to 60°C, which is lethal for the yeast. After the decrease in yeast, acetic acid accumulated to levels as high as 20mg/g-ds in the composting material and vigorous organic matter degradation was inhibited. However, by maintaining the temperature at 40°C for 2days during the heating phase in the early stage of composting, both the organic acids originally contained in the raw material and acetic acid produced during the heating phase were degraded by the yeast. The concentration of acetic acid was kept at a relatively low level (10.1mg/g-ds at the highest), thereby promoting the degradation of organic matter by other microorganisms and accelerating the composting process. These results indicate that temperature control enhances the effects of microbial inoculation into composts.

Direct production of ethanol from neoagarobiose using recombinant yeast that secretes α-neoagarooligosaccharide hydrolase.

An α-neoagarooligosaccharide hydrolase, AgaNash, was purified from Cellvibrio sp. OA-2007, which utilizes agarose as a substrate. The agaNash gene, which encodes AgaNash, was obtained by comparing the N-terminal amino acid sequence of AgaNash with that deduced from the nucleotide sequence of the full-length OA-2007 genome. The agaNash gene combined with the Saccharomyces cerevisiae signal peptide α-mating factor was transformed into the YPH499 strain of S. cerevisiae to generate YPH499/pTEF-MF-agaNash, and the recombinant yeast was confirmed to produce AgaNash, though it was mainly retained within the recombinant cell. To enhance AgaNash secretion from the cell, the signal peptide was replaced with a combination of the signal peptide and a threonine- and serine-rich tract (T-S region) of the S. diastaticus STA1 gene. The new recombinant yeast, YPH499/pTEF-STA1SP-agaNash, was demonstrated to secrete AgaNash and hydrolyze neoagarobiose with an efficiency of as high as 84%, thereby producing galactose, which is a fermentable sugar for the yeast, and ethanol, at concentrations of up to 1.8 g/L, directly from neoagarobiose.